PROJECT SUMMARY/ABSTRACT Neoantigens have been identified by us and others as important targets of immunotherapy in the context of checkpoint blockade therapy, adoptive cell therapy, and vaccine therapy. We have recently initiated two phase 1 clinical trials of first generation neoantigen vaccines in breast cancer based on the neoantigen DNA vaccine and synthetic long peptide vaccine platforms. In preclinical models, we have optimized sequencing and epitope prediction algorithms for the identification and prioritization of neoantigens, and we have leveraged innovative technologies (CyTOF, single cell RNA sequencing) to comprehensively interrogate the immune response to neoantigen vaccination and other cancer immunotherapies. We propose to build on these observations by performing a randomized phase 1 trial of neoantigen DNA vaccine alone vs. neoantigen DNA vaccine plus anti-PD-L1, in patients with persistent triple negative breast cancer (TNBC) following neoadjuvant chemotherapy. The overall hypothesis is that enhancing neoantigen-specific T cell responses can improve clinical outcomes in TNBC. This hypothesis will be tested by completing the following aims: Specific Aim 1. Test the hypothesis that neoantigen vaccines +/- anti-PD-L1 can induce and/or enhance neoantigen-specific T cell responses. We have recently initiated a randomized phase 1 clinical trial of neoantigen DNA vaccines +/- anti-PD-L1 in TNBC patients with persistent disease following neoadjuvant chemotherapy. Next-generation sequencing and epitope prediction algorithms will be used to prioritize neoantigens. Neoantigen DNA vaccines will be designed and manufactured in the GMP facility at SCC. Specific Aim 2: Test the hypothesis that targeting tumor-associated macrophages (TAM) can enhance neoantigen-specific T cell responses in TNBC. Both embryonically-derived tissue-resident TAM and inflammatory monocyte-derived TAM restrain antitumor immunity in TNBC. We propose to combine CSF1/CSF1R blockade with CCR2 inhibition to simultaneously target both sources of TAM in the context of neoantigen vaccination in two mouse breast cancer models. The tumor microenvironment will be characterized by CyTOF and single cell RNA sequencing. Specific Aim 3: Test the hypothesis that a neoantigen simian Ad vector vaccine prime followed by a neoantigen DNA vaccine boost can enhance the response to breast cancer neoantigens. We propose a prime/boost strategy consisting of a simian recombinant Ad vaccine prime, followed by a plasmid DNA vaccine boost. This prime/boost neoantigen vaccine strategy represents a state-of-the-art vaccine strategy to more effectively induce antitumor immunity, and leverages expertise at WUSM within the fields of neoantigen DNA vaccines and recombinant Ad vector vaccines. Photocleavable tetramer analysis, CyTOF, and single cell RNA sequencing will be performed on neoantigen-specific T cells in the peripheral blood and the tumor microenvironment to rigorously evaluate the neoantigen-specific T cell response to vaccination.